1. Field of the Invention
This invention relates to biological material. More particularly, this invention relates to an immobilized biological material (an immobilized biologically active material) having at least one primary or secondary amino group per molecule and to a process for immobilizing such material. Even more particularly, this invention relates to: (a) such biological material which has been immobilized by contacting it with an excess of an isocyanate-capped liquid polyurethane prepolymer to form a mixture which is cured by contacting it (the mixture) with an amount of a curing agent (e.g., water or an appropriate amine) effective for curing the biological material; and (b) to the process whereby such biological material is immobilized.
2. Description of the Prior Art
A review of enzyme technology was published in the Aug. 18, 1975 issue of Chemical & Engineering News (pp. 22-41). Chemical & Engineering News is published by the American Chemical Society.
U.S. Pat. No. 3,574,062 (195/63, Sato) teaches a method for preparing a bound protein (an enzyme) wherein a polyester polyurethane is diazotized with a diazonium salt of an amino acid and then coupled with a nonenzymatic animal protein to form a diazotized polyurethane which is reacted with an enzyme to form the immobilized enzyme.
U.S. Pat. No. 3,705,084 (195/63, Reynolds) teaches a a flow-through enzyme reactor comprising: (a) a macroporous reactor core; (b) a polymeric surface (which can be a polyurethane resin) on the reactor core; (c) an enzyme adsorbed on the polymeric surface and cross-linked in place thereon by a difunctional agent (e.g., a polyisocyanate).
Reynolds prepares the immobilized enzyme for his reactor by adsorbing an active enzyme on a polymeric surface and further immobilizing the enzyme by crosslinking it in place with a crosslinking agent such as a monomeric polyisocyanate.
German Offenlegungsschrift No. 2,319,706 published Nov. 15, 1973 teaches an enzyme bound to a polyurethane foam and a method for preparing such bound enzyme.
U.S. Pat. No. 3,791,927 (195/63, Forgione et al) teaches a water-insoluble bound protein (enzyme) entrapped within the cells of a self-supporting reticulated cellular material (which can be a polyurethane foam), the protein (enzyme) being bound to the cellular material.
U.S. Pat. No. 3,672,955 (195/68, Stanley) teaches a process for preparing a bound protein (enzyme) comprising: (a) emulsifying an aqueous dispersion of the enzyme with a solution of a polyisocyanate in a volatile water-immiscible solvent (e.g., methylchloroform); (b) admixing the resulting emulsion with a solid particulate carrier; and (c) evaporating the solvent therefrom. Stanley's polyisocyanate can be an isocyanate-capped liquid polyurethane prepolymer. Said U.S. Pat. No. 3,672,955, in its entirety, is incorporated herein by reference.
It is noted that, in his Example 3, Stanley reports the binding of an enzyme component (a peroxidase) of a fermentation broth by admixing a portion of the broth with a polyisocyanate dissolved in methylchloroform. It seems probably that, under Stanley's reaction conditions, any other enzymes which were present in the broth plus any coenzymes which contained a primary or secondary amino group and which were present in the broth would have been immobilized (rendered insoluble).
Silman et al, Annual Review of Biochemistry, 1966, 35 (Part 2), pages 873-908 present a review of methods for preparing water-insoluble derivatives of enzymes, antigens, and antibodies.
Singer, Nature, 1959, 183, 1523-1524 teaches a method for reacting a protein with a diisocyanate (m-xylene diisocyanate).
U.S. Patent Application Ser. No. 250,012, filed May 3, 1972, and now abandoned (Wood et al, inventors) which is assigned to W. R. Grace & Co. teaches, in Example 21, a foamed polyurethane comprising an immobilized enzyme (urease), a method for preparing such immobilized enzyme, and a method for using it.
Said Application Ser. No. 250,012 also teaches, e.g., in claim 8, a foamable composition comprising: (a) an isocyanate-capped polyurethane prepolymer; (b) water; and (c) biostats, fungicides, or enzymes. A similar teaching occurs in claim 7 of the above-mentioned German Offenlegungsschrift No. 2,319,706.
U.S. Patent Application Ser. No. 362,488, filed May 21, 1973, and now abandoned (Wood et al, inventors) which is assigned to W. R. Grace & Co. teaches the preparation of a bound protein (an enzyme) by a process comprising contacting an isocyanate-capped liquid polyurethane prepolymer with an aqueous dispersion of the enzyme under foam-forming conditions, whereby the polyurethane foams and the enzyme becomes integrally bound to the resulting polyurethane foam.
It is noted that, in said Application Ser. No. 362,488, Wood et al report, in their Example 1, that an enzyme (cellulase) present in a fermentation broth was immobilized (bound or rendered insoluble) by admixing the broth with an isocyanate-capped liquid polyurethane prepolymer under conditions which produced a foam. It seems probable that, under the conditions of said Example 3, any other enzymes present in the broth plus any coenzymes which contained a primary or secondary amino groups and which were present in the broth would have been immobilized.
Dean et al, U.S. Pat. No. 3,904,478, teach a method for immobilizing coenzymes wherein the coenzyme is immobilized by: (a) forming a thio derivative of the coenzyme in which an amino group of the coenzyme is replaced by a thio group; (b) reacting the thio group with: (i) a bifunctional organic compound having a nucleophilic group so as to couple the bifunctional organic compound to the coenzyme and then coupling the resultant coenzyme reaction product to a water insoluble support material through the unreacted functional group of the bifunctional organic compound; or (ii) a water insoluble support material having a plurality of pendant nucleophilic groups so as to couple the coenzyme to the support material.
Immobilized coenzymes are also taught by: (a) Mosbach et al, Enzyme Eng., 2nd, 1973 (published in 1974) which is abstracted in Chemical Abstracts, 1975, 83, 39185c; (b) McCormick, Methods Enzymol., 1974, 34(Affinity Tech.; Enzyme Purif. Part B), 300-302, which is abstracted in Chemical Abstracts, 1975, 83, 24325z; (c) Barker, Methods Enzymol., 1974, 34(Affinity Tech.; Enzyme Purif., Part B), 479-491, which is abstracted in Chemical Abstracts 1975, 83, 39459v; (d) Morse et al, U.S. Pat. No. 3,860,733, which is abstracted in Chemical Abstracts 1975, 82, 138021g; (e) French Patent No. 2,206,129 (to Merck and Co., Inc.), which is abstracted in Chemical Abstracts 1975, 82, 138024k; (f) Chibata et al, Enzyme Eng., 2nd, 1973 (published 1974), 229-236, which is abstracted in Chemical Abstracts 1975, 83, 39356j; (g) Weibel et al, Enzyme Eng., 2nd, 1973 (published 1974) 203-208, which is abstracted in Chemical Abstracts 1975, 83, 55279s; and (h) Molteni et al, Eur. J. Med. Chem.-Chim. Ther. 1974, 9(6), 618-620, which is abstracted in Chemical Abstracts 1975, 83, 48130h.
U.S. Pat. No. 2,781,339 (260/211.5, Mitz et al) teaches a process for preparing a coenzyme (coenzyme A) comprising admixing a crude aqueous solution of the coenzyme with acid-conditioned charcoal to absorb the coenzyme and thereafter eluting the coenzyme from the charcoal.
Immobilized antibiotics are taught by: (a) Wagman et al, Antimicrob. Agents Chemother, 1975, 7(3), 316-319, which is abstracted in Chemical Abstracts 1975, 83, 22169c; and (b) Seela, Z. Naturforsch., C: Biosci., 1975, 30c(7-8), 544-545, which is abstracted in Chemical Abstracts 1975, 83, 74898j.
U.S. Pat. No. 3,905,923 (260/2.5 AD, Klug) teaches an immobilized enzyme system formed from an enzyme and a hydrophilic poly(urea-urethane) foam, the foam surrounding, entrapping, and supporting the enzyme in an active configuration. The hydrophilic foam is formed by the reaction of water with a hydrophilic isocyanate-capped polyoxyalkylene prepolymer.
Isocyanate-capped polyurethane prepolymers are well known to those skilled in the art. See, for example: (a) the penultimate paragraph on page 854 of Volume 9 of the Second Edition of the Kirk-Othmer "Encyclopedia of Chemical Technology", John Wiley and Sons, Inc., New York, N.Y.; or (b) the third full paragraph in the left hand (first) column of page 872 of the Second Edition of "The Encyclopedia of Chemistry" , George L. Clark, Editor, Reinhold Publishing Corporation, New York, N.Y.
It is noted that the cured isocyanate-capped liquid polyurethane prepolymers of our invention (including those comprising immobilized biological materials (i.e., immobilized biologically active materials which are also referred to herein as "immobilized group member(s)") produced by foaming an isocyanate-capped liquid polyurethane prepolymer with water or by the reaction of said prepolymer with water under nonfoaming conditions comprise poly(urea-urethane) products which may be foams, films, discs, tubes, rods, spheres, or the like, and which can be hydrophilic.